Bulked-Segregant Analysis Coupled to Whole Genome Sequencing (BSA-Seq) for Rapid Gene Cloning in Maize

Forward genetics remains a powerful method for revealing the genes underpinning organismal form and function, and for revealing how these genes are tied together in gene networks. In maize, forward genetics has been tremendously successful, but the size and complexity of the maize genome made identifying mutant genes an often arduous process with traditional methods. The next generation sequencing revolution has allowed for the gene cloning process to be significantly accelerated in many organisms, even when genomes are large and complex. Here, we describe a bulked-segregant analysis sequencing (BSA-Seq) protocol for cloning mutant genes in maize. Our simple strategy can be used to quickly identify a mapping interval and candidate single nucleotide polymorphisms (SNPs) from whole genome sequencing of pooled F2 individuals. We employed this strategy to identify narrow odd dwarf as an enhancer of teosinte branched1, and to identify a new allele of defective kernel1. Our method provides a quick, simple way to clone genes in maize

The Regulatory Landscape of a Core Maize Domestication Module Controlling Bud Dormancy and Growth Repression

Many domesticated crop plants have been bred for increased apical dominance, displaying greatly reduced axillary branching compared to their wild ancestors. In maize, this was achieved through selection for a gain-of-function allele of the TCP transcription factor teosinte branched1 (tb1). The mechanism for how a dominant Tb1 allele increased apical dominance, is unknown. Through ChIP seq, RNA seq, hormone and sugar measurements on 1 mm axillary bud tissue, we identify the genetic pathways putatively regulated by TB1. These include pathways regulating phytohormones such as gibberellins, abscisic acid and jasmonic acid, but surprisingly, not auxin. In addition, metabolites involved in sugar sensing such as trehalose 6-phosphate were increased. This suggests that TB1 induces bud suppression through the production of inhibitory phytohormones and by reducing sugar levels and energy balance. Interestingly, TB1 also putatively targets several other domestication loci, including teosinte glume architecture1, prol1.1/grassy tillers1, as well as itself. This places tb1 on top of the domestication hierarchy, demonstrating its critical importance during the domestication of maize from teosinte

Altered expression of maize PLASTOCHRON1 enhances biomass and seed yield by extending cell division duration

Maize is the highest yielding cereal crop grown worldwide for grain or silage. Here, we show that modulating the expression of the maize PLASTOCHRON1 (ZmPLA1) gene, encoding a cytochrome P450 (CYP78A1), results in increased organ growth, seedling vigour, stover biomass and seed yield. The engineered trait is robust as it improves yield in an inbred as well as in a panel of hybrids, at several locations and over multiple seasons in the field. Transcriptome studies, hormone measurements and the expression of the auxin responsive DR5(rev): mRFPer marker suggest that PLA1 may function through an increase in auxin. Detailed analysis of growth over time demonstrates that PLA1 stimulates the duration of leaf elongation by maintaining dividing cells in a proliferative, undifferentiated state for a longer period of time. The prolonged duration of growth also compensates for growth rate reduction caused by abiotic stresses

Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein–Protein Interactions

Protein–protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PIL) and AP3-like (AP3L) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PIL protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PIL homodimerization is an anomaly or indicative of broader trends, we characterized PIL dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PIL homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PIL homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PIL protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PIL dimerization activity. Furthermore, ectopic expression of a Joinvillea PIL homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner. Key words: PISTILLATA, Poales, APETALA3, convergent molecular evolution, B-class MADS box genes, evolution of flower development

Boundary domain genes were recruited to suppress bract growth and promote branching in maize

Grass inflorescence development is diverse and complex and involves sophisticated but poorly understood interactions of genes regulating branch determinacy and leaf growth. Here, we use a combination of transcript profiling and genetic and phylogenetic analyses to investigate tasselsheath1 (tsh1) and tsh4, two maize genes that simultaneously suppress inflorescence leaf growth and promote branching. We identify a regulatory network of inflorescence leaf suppression that involves the phase change gene tsh4 upstream of tsh1 and the ligule identity gene liguleless2 (lg2). We also find that a series of duplications in the tsh1 gene lineage facilitated its shift from boundary domain in nongrasses to suppressed inflorescence leaves of grasses. Collectively, these results suggest that the boundary domain genes tsh1 and lg2 were recruited to inflorescence leaves where they suppress growth and regulate a nonautonomous signaling center that promotes inflorescence branching, an important component of yield in cereal grasses

Wildflowers of the Mountain West Wildflowers of the Mountain West Richard M. Anderson, JayDee Gunnell, Jerry L. Goodspeed, 2012 Utah State University Press, an imprint of University Press of Colorado Boulder, CO. 300 pp. ISBN: 978-0-87421-895-4 (spiral bo

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B and C class MADS-box genes and the developmental genetics of maize flower development

The ABC model of flower development describes how a flower is patterned and the genes necessary for floral organ identity. However, it is not clear that the ABC model can be generally applied to the flowering plants, as it was based solely on genetic studies from the core eudicot species Arabidopsis and Antirrhinum. This dissertation describes an examination of maize orthologs of B and C class genes, and compares their function with B and C class genes of Arabidopsis to understand the degree to which the ABC model is conserved. B class genes from maize were found to rescue Arabidopsis B class mutants, and the maize B class proteins were shown to bind DNA as an obligate heterodimer as has been demonstrated in Arabidopsis. These findings indicate conservation in biochemical function of the maize and Arabidopsis B class proteins. Furthermore, these findings support the conclusion that the lodicule, a grass specific organ of uncertain homology, represents a modified petal. A comparative expression approach was used to further verify the relationship of lodicules to the organs of non-grass flowers. B class genes were shown to be expressed in a whorl of foliar organs outside the stamens in Streptochaeta, a basal grass that diverged before the evolution of lodicules, and in the petals of the outgroup species Joinvillea and Chondropetalum strongly supporting the interpretation that lodicules are modified petals, and further supporting conservation of B class function between Arabidopsis and maize. Zag1 and Zmm2 are duplicate pair of C class genes from maize that are hypothesized to have partitioned the C class function of establishing stamen and carpel identity. Rescue of the Arabidopsis C class mutant ag with the two maize genes confirms that their protein products have subfunctionalized, with ZAG1 better able to promote carpel identity, and ZMM2 better able to promote stamen identity. A more recent duplicate of Zmm2 was isolated, Zmm23, as were mutant alleles of zmm2 and zmm23. While the zmm2 zmm23 double mutant had no phenotype, the zag1 zmm2 zmm23 showed a considerable enhancement of the previously described zag1 phenotype substantiating a C class function for Zmm2 and Zmm2

Protein change in plant evolution: tracing one thread connecting molecular and phenotypic diversity

Proteins change over the course of evolutionary time. New protein-coding genes and gene families emerge and diversify, ultimately affecting an organism’s phenotype and interactions with its environment. Here we survey the range of structural protein change observed in plants and review the role these changes have had in the evolution of plant form and function. Verified examples tying evolutionary change in protein structure to phenotypic change remain scarce. We will review the existing examples, as well as draw from investigations into domestication, and quantitative trait locus (QTL) cloning studies searching for the molecular underpinnings of natural variation. The evolutionary significance of many cloned QTL has not been assessed, but all the examples identified so far have begun to reveal the extent of protein structural diversity tolerated in natural systems. This molecular (and phenotypic) diversity could come to represent part of natural selection’s source material in the adaptive evolution of novel traits. Protein structure and function can change in many distinct ways, but the changes we identified in studies of natural diversity and protein evolution were predicted to fall primarily into one of six categories: altered active and binding sites; hypomorphic and hypermorphic alleles; altered protein-protein interactions; altered domain content; altered protein stability; and altered activity as an activator or repressor. Variability was also observed in the evolutionary scale at which particular changes were observed. Some changes were detected at both micro- and macroevolutionary timescales, while others were observed primarily at deep or shallow phylogenetic levels. This variation might be used to determine the trajectory of future investigations in structural molecular evolution